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  1. Science & Research (Biologics)

Influenza neuraminidase antigenicity and efficacy in vaccines


Robert Daniels

Robert Daniels, PhD

Office of Vaccines Research and Review
Division of Viral Products
Laboratory of Pediatric and Respiratory Viral Disease



Dr. Robert Daniels received his PhD in molecular and cellular biology from the University of Massachusetts, Amherst in 2007 and completed post-doctoral training at the Karolinska Institute in Sweden. In 2010 he became an assistant professor in the Department of Biochemistry and Biophysics at Stockholm University where he used his expertise in biochemistry and secretory protein folding to establish a research group that examined the maturation and evolution of the influenza virus surface antigen, neuraminidase (NA). He also received the 2018 Stockholm University teacher of the year award. In 2019, Dr. Daniels joined the Laboratory of Pediatric and Respiratory Viral Diseases (LPRVD) in the Division of Viral Products (DVP), in the Office of Vaccine Research and Review (OVRR) at CBER. His group focuses on increasing influenza vaccine efficacy and cross protection by developing methodologies for improving the response against the NA antigen in vaccines and for assessing NA in circulating strains and vaccine preparations.

General Overview

Influenza viruses are estimated to cause symptomatic infections in 3-11% of the U.S. population annually and severe disease in about 1.5% of those infected. Although several drugs now available can limit the severity of an influenza infection, yearly vaccination remains the most effective approach to reduce the disease burden caused by influenza viruses.

Current influenza vaccines include split inactivated influenza viruses, live attenuated influenza viruses, and recombinant hemagglutinin (HA) antigens. Each vaccine type has advantages and all of them protect against the two influenza A subtypes (H1N1 and H3N2) and at least one of the influenza B lineages (Yamagata and Victoria) that are responsible for seasonal infections in humans.

Manufacturing split inactivated influenza vaccines generally involves propagating candidate vaccine viruses (CVVs) in eggs or mammalian cells, whereas recombinant HA vaccines are produced using insect cells. Despite these differences, both products are standardized based on the HA antigen content, as responses against HA correlate well with protection.

Each season, several inter-connected challenges can affect the influenza vaccine efficacy: 1) Influenza viruses are constantly evolving, which  can cause antigenic drift and occasional antigenic shift in type A viruses; 2) vaccine strains must be selected months in advance to meet manufacturing deadlines; 3) viral propagation in eggs or cells can lead to unexpected adaptations that can alter important antigens in the vaccine.

Although influenza vaccines have mainly been developed to generate an optimal immune response against HA, influenza viruses do possess a second, less abundant surface antigen, neuraminidase (NA). Like HA, antibodies that recognize NA can provide both matched and cross-protection against influenza virus strains. NA also evolves and drifts independently of HA. These properties imply that by improving the NA response, it might be possible to increase the breadth of the vaccine coverage and mitigate many of the yearly challenges that influenza vaccines face.

In the split inactivated and the live attenuated influenza virus vaccines, NA is present. However, many technical issues must first be solved before the NA component of the annual vaccines can be regulated. Our laboratory is systematically addressing several of these issues to establish a framework for improving the ability of NA to increase the breadth and efficacy of the annual vaccine.

Scientific Overview


The main goal of the lab is to increase the vaccine efficacy and cross-protection against influenza viruses by improving the NA antigen response. The illustration depicts the analytical and experimental approaches that the lab utilizes in an effort to enhance the NA response elicited by viral and protein-based influenza vaccines.

Influenza viruses contain two surface antigens, the receptor-binding protein, hemagglutinin (HA), and the receptor-destroying enzyme, neuraminidase (NA). However, influenza vaccines have primarily been developed using methodologies centered on the more abundant HA antigen. Our long-term objectives are to increase the breadth and efficacy of the annual influenza vaccine by establishing a similar framework for assessing the NA antigen.

To reach this goal, we are working to create methods capable of rapidly monitoring changes in NA antigenicity and to define the NA parameters that correlate with protection. We will use these parameters to assess the immunogenic NA content during the vaccine manufacturing process and to determine if the quantity is sufficient. In parallel, we are developing approaches to increase the NA content in CVVs, retain its immunogenicity throughout the different manufacturing processes, and to rationally design NAs for improved immunogenicity in recombinant-based vaccines.

Within the lab, these objectives are separated into the following research areas: 1) NA assay development for characterizing circulating strains; 2) engineering CVVs to increase NA responses from viral-based vaccines; 3) rationally designing recombinant NAs for improved production and immunogenicity.

We address each research area using a similar systematic approach that generally involves in vitro biochemical analysis followed by validation tests that include cell-based assays and in vivo animal models. The techniques we utilize include: enzyme kinetics, protein/viral purification, analytical assays, viral reverse genetics with propagation in cells and eggs, and viral immunization and challenge models. This broad range of approaches are carried out using the most up to date equipment and techniques so that advancements in one area of vaccine manufacturing can be rapidly assessed in another.

The results from this work will likely help to establish the frameworks that are necessary to better utilize the NA antigen in the influenza vaccine and to identify the NAs in circulating strains that will provide the greatest breadth of coverage for upcoming seasons. Together, these concepts should help to advance influenza vaccine manufacturing and to improve the efficacy of the yearly vaccine.


  1. Vaccine 2024 Jan 25;42(3):608-19
    Vaccine-associated respiratory pathology correlates with viral clearance and protective immunity after immunization with self-amplifying RNA expressing the spike (S) protein of SARS-CoV-2 in mouse models.
    Kachko A, Selvaraj P, Liu S, Kim J, Rotstein D, Stauft CB, Chabot S, Rajasagi N, Zhao Y, Wang T, Major M
  2. J Chromatogr B 2024 Jan;1232:123975
    Isolation by multistep chromatography improves the consistency of secreted recombinant influenza neuraminidase antigens.
    Kang H, Malik T, Daniels R
  3. Vaccine 2023 Jun 29;41(29):4302-12
    Inactivated influenza virions are a flexible vaccine platform for eliciting protective antibody responses against neuraminidase.
    Martinez MR, Gao J, Wan H, Kang H, Klenow L, Daniels R
  4. Nano Lett 2023 Apr 26;23(8):3377-84
    Thermoplasmonic vesicle fusion reveals membrane phase segregation of influenza spike proteins.
    Moreno-Pescador G, Arastoo MR, Ruhoff VT, Chiantia S, Daniels R, Bendix PM
  5. ACS Synth Biol 2023 Feb 17;12(2):432-45
    Silencing transcription from an influenza reverse genetics plasmid in E. coli enhances gene stability.
    Malik T, Klenow L, Karyolaimos A, Gier JW, Daniels R
  6. J Biol Chem 2023 Feb;299(2):102891
    Influenza virus and pneumococcal neuraminidases enhance catalysis by similar yet distinct sialic acid-binding strategies.
    Klenow L, Elfageih R, Gao J, Wan H, Withers SG, de Gier JW, Daniels R
  7. ACS Infect Dis 2023 Jan 13;9(1):33-41
    Sialosides containing 7-N-acetyl sialic acid are selective substrates for neuraminidases from influenza A viruses.
    Kooner AS, Yuan Y, Yu H, Kang H, Klenow L, Daniels R, Chen X
  8. Nat Commun 2022 Dec 21;13(1):7864
    Antibodies targeting the neuraminidase active site inhibit influenza H3N2 viruses with an S245N glycosylation site.
    Stadlbauer D, McMahon M, Turner HL, Zhu X, Wan H, Carreño JM, O'Dell G, Strohmeier S, Khalil Z, Luksza M, van Bakel H, Simon V, Ellebedy AH, Wilson IA, Ward AB, Krammer F
  9. NPJ Vaccines 2022 Jul 14;7(1):79
    Antigenic comparison of the neuraminidases from recent influenza A vaccine viruses and 2019-2020 circulating strains.
    Gao J, Li X, Klenow L, Malik T, Wan H, Ye Z, Daniels R
  10. PLoS One 2022 May 26;17(5):e0268924
    Involvement of a putative ATP-binding cassette (ABC) involved in manganese transport in virulence of Listeria monocytogenes.
    Liu Y, Yoo BB, Hwang CA, Martinez MR, Datta AR, Fratamico PM
  11. J Virol 2021 Nov;95(24):e0116021
    Design of the recombinant influenza neuraminidase antigen is crucial for its biochemical properties and protective efficacy.
    Gao J, Klenow L, Parsons L, Malik T, Phue JN, Gao Z, Withers SG, Cipollo J, Daniels R, Wan H
  12. PLoS Pathog 2021 Apr 19;17(4):e1009171
    Balancing the influenza neuraminidase and hemagglutinin responses by exchanging the vaccine virus backbone.
    Gao J, Wan H, Li X, Rakic Martinez M, Klenow L, Gao Y, Ye Z, Daniels R
  13. Biophys J 2021 Feb 12;120(3 Suppl. 1):48a
    Phase partitioning of influenza virus neuraminidase in a model membrane.
    Arastoo MR, Pescador GSM, Veje EL, Ostbye H, Daniels R, Bendix PM
  14. J Virol 2020 Oct;94(19):e00874-20
    N-linked glycan sites on the influenza NA head domain are required for efficient IAV incorporation and replication.
    Östbye H, Gao J, Rakic Martinez M, Wang H, de Gier JW, Daniels R
  15. mSphere 2020 Apr;5(2):e00187-20
    In support of simian polyomavirus 40 VP4 as a later expressed viroporin.
    Daniels R, Hebert D
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